Human tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) is a severe global health problem, responsible for approx. 3 million deaths annually, according to the WHO. The worldwide incidence of new tuberculosis (TB) cases had been falling during the 1960s and 1970s but during recent years this trend has markedly changed in part due to the advent of AIDS and the appearance of multidrug resistant strains of M. tuberculosis. 
The only vaccine presently available for clinical use is BCG, a vaccine whose efficacy remains a matter of controversy. BCG generally induces a high level of acquired resistance in animal models of TB, but several human trials in developing countries have failed to demonstrate significant protection. Notably, BCG is not approved by the FDA for use in the United States because BCG vaccination impairs the specificity of the Tuberculin skin test for diagnosis of TB infection.
This makes the development of a new and improved vaccine against TB an urgent matter, which has been given a very high priority by the WHO. Many attempts to define protective mycobacterial substances have been made, and different investigators have reported increased resistance after experimental vaccination. However, the demonstration of a specific long-term protective immune response with the potency of BCG has not yet been achieved.
Immunity to M. tuberculosis is characterized by some basic features; specifically sensitized T lymphocytes mediates protection, and the most important mediator molecule seems to be interferon gamma (IFN-γ).
M. tuberculosis holds, as well as secretes, several proteins of potential relevance for the generation of a new TB vaccine. For a number of years, a major effort has been put into the identification of new protective antigens for the development of a novel vaccine against TB. The search for candidate molecules has primarily focused on proteins released from dividing bacteria. Despite the characterization of a large number of such proteins only a few of these have been demonstrated to induce a protective immune response as subunit vaccines in animal models, most notably ESAT-6 and Ag85B (Brandt et al 2000 Infect. Imm. 68:2; 791-795).
In 1998 Cole et al published the complete genome sequence of M. tuberculosis and predicted the presence of approximately 4000 open reading frames (Cole et al 1998). Following the sequencing of the M. tuberculosis genome, nucleotide sequences comprising Rv0288, Rv3019c or Rv3017c are described in various databases and putative protein sequences for the above sequences are suggested, Rv3017c either comprising methionin or valine as the first amino acid (The Sanger Centre database (http://www.sanger.ac.uk/Projects/M—tuberculosis), Institut Pasteur database (http://genolist.pasteur.fr/TubercuList) and GenBank (http://www4.ncbi.nlm.nih.gov)). However important, this sequence information cannot be used to predict if the DNA is translated and expressed as proteins in vivo. More importantly, it is not possible on the basis of the sequences, to predict whether a given sequence will encode an immunogenic or an inactive protein. The only way to determine if a protein is recognized by the immune system during or after an infection with M. tuberculosis is to produce the given protein and test it in an appropriate assay as described herein.
Diagnosing M. tuberculosis infection in its earliest stage is important for effective treatment of the disease. Current diagnostic assays to determine M. tuberculosis infection are expensive and labour-intensive. In the industrialised part of the world the majority of patients exposed to M. tuberculosis receive chest x-rays and attempts are made to culture the bacterium in vitro from sputum samples. X-rays are insensitive as a diagnostic assay and can only identify infections in a very progressed stage. Culturing of M. tuberculosis is also not ideal as a diagnostic tool, since the bacteria grows poorly and slowly outside the body, which can produce false negative test results and take weeks before results are obtained. The standard tuberculin skin test is an inexpensive assay, used in third world countries, however it is far from ideal in detecting infection because it cannot distinguish M. tuberculosis-infected individuals from M. bovis BCG-vaccinated individuals and therefore cannot be used in areas of the world where patients receive or have received childhood vaccination with bacterial strains related to M. tuberculosis, e.g. a BCG vaccination.
Animal tuberculosis is caused by Mycobacterium bovis, which is closely related to M. tuberculosis and within the tuberculosis complex. M. bovis is an important pathogen that can infect a range of hosts, including cattle and humans. Tuberculosis in cattle is a major cause of economic loss and represents a significant cause of zoonotic infection. A number of strategies have been employed against bovine TB, but the approach has generally been based on government-organised programmes by which animals deemed positive to defined screening test are slaughtered. The most common test used in cattle is Delayed-type hypersensitivity with PPD as antigen, but alternative in vitro assays are also developed. However, investigations have shown the both the in vivo and the in vitro tests have a relative low specificity, and the detection of false-positive is a significant economic problem (Pollock et al 2000). There is therefore a great need for a more specific diagnostic reagent, which can be used either in vivo or in vitro to detect M. bovis infections in animals.